Quantitative comparison of PI(3,5)P₂ biosensors reveals SnxA is the most sensitive and unbiased

PI(3,5)P₂ is an endosomal lipid whose depletion is associated with a variety of pathologies such as neurodegenerative diseases. However, studying this lipid in physiological and disease models has been difficult due to the scarcity of the lipid and the lack of live-cell imaging tools. That is, until recently, when a novel PI(3,5)P₂ biosensor, SnxA, was characterized. Despite the exciting promise of this new sensor, it was still unclear whether SnxA reported on PI(3,5)P₂ levels without bias and how its sensitivity compared with other PI(3,5)P₂ biosensors. In this work, we addressed these gaps by using a recruitable PIKfyve construct to demonstrate that ectopically generated PI(3,5)P₂ at mitochondria was sufficient to recruit SnxA. Further, we co-expressed putative PI(3,5)P₂ biosensors to definitively show that SnxA is more sensitive to PI(3,5)P₂. We also validated previous results by showing that SnxA depends on PI(3,5)P₂ for membrane binding, that SnxA responds to PI(3,5)P₂ production at endosomes, and that PI(3,5)P₂ levels decline quickly when its production is inhibited. Thus, we conclude that SnxA is a robust and sensitive PI(3,5)P₂ biosensor that facilitates real-time analysis of this key lipid.